Fermentation broth degassification

ABSTRACT

A post-fermentation degassing technique is provided which is directed for use in degassing foamed fermentation broths containing cultured microorganisms. Such a broth is charged while pressurized first through a heat exchanger to cool same, and then through a nozzle to form a spray suspension in a gaseous spray chamber located in a gravitationally upper part of a degassing vessel. The spray chamber is vented to the atmosphere. The spray suspension is coalesced to form a bulk liquid phase located in a gravitationally lower portion of the degassing vessel whereby the unpon surface of such bulk liquid phase forms the buttom surface of such spray chamber. The nozzle is so oriented that the spray suspension therefrom is downwardly directed towards the surface of the bulk liquid phase and the layer of foam characteristically formed thereon.

This application is a division under 37, C.F.R. 1.60 of application Ser.No. 181,452, filed Apr. 14, 1988 now U.S. Pat. No. 4,970,167, issuedNov. 13, 1990.

BACKGROUND

1. Field of the Invention

This invention lies in the field of processes and apparatus forproducing degassed fermentation broth.

2. Prior Art

Fermentors which are pressurizable and which are capable of using highair flow are known, as is technology for culturing microorganisms insuch a fermentor. A pressurized culture zone with high air flow isdesirable because it improves the rate of oxygen transfer between themicroorganisms and the aqueous culturing medium, increases the rate ofmicroorganism growth, and increases the density of microorganismscultured, but it also increases the foam inherently produced, as thoseskilled in the art appreciate.

The foam causes a significant problem in post-fermentation separation ofthe fermentation broth liquid phase from the gas phase which is in theform of entrained gas bubbles (foam). This problem is exacerbated whencontinuous pressurized fermentation procedures are practiced with highair flow.

The foaming inherently occurs during fermentation, especially whenconducted under pressure and high air flow, because the air (oxygen)charged into the fermentor becomes admixed with the aqueous culturemedium under the action of medium agitation which is characteristicallyemployed to maintain the medium in a uniform state and to promotetransfer of oxygen into microorganisms being aerobically cultured.Certain agents present in the culture medium which originate both fromthe nutrients charged into the culture medium and from the metabolitesexcreted from the microorganisms being cultured exert significantsurfactant activity and thus tend to create and to stabilize foaming.

Defoaming agents are undesirable additives to a fermentation broth whenthe fermentation product is intended for food use since they contaminatethe broth. Also, the level at which such agents would need to be addedin the case of a broth containing a relatively high level of totalsolids (such as exists when pressurized fermentation is practiced) inorder to achieve a practical level of defoaming is so great as to causeoperating cost problems and final product purity problems (for example,as regards the level of impurities permitted by the U.S. Food and DrugAdministration in human food).

Various mechanical defoaming techniques for use in a fermentor areknown, such as rotable metal arm arrangements adapted for flailing thesurface regions of a foam, centrifuging, and the like. For example, HuntU.S. Pat. No. 4,373,024 describes a rotable foam breaking apparatuswhich is mounted in the upper central portion of a fermentor. When thisapparatus functions in an operating fermentor, a foamed fermentationbroth is drawn up thereinto along a multitude of paths. At or near thepath rotational axes, the gas phase tends to be separated from theliquid phase by centrifugal force, and the latter phase is returned tothe main mass of the fermentation broth while the former phase isseparated and is vented.

Unfortunately, the rate at which any known apparatus is able to separatethe gas phase from the liquid phase in a fermentor seems to be generallyinsufficient to meet the gas separation requirements existing in highpressure, high air flow continuous fermentation so that such apparatusdoes not solve the post-fermentation gas separation problem.

In the prior art the post fermentation gas separation problem wastypically previously solved by removing the gas-filled fermentationbroth from the fermentor and passing such into tin open holdingreservoir for holding until the gas bubbles collapse. However, the rateof foam collapse is slow, the quantity of gas is large, and thestability of foam is variable from one microorganism to another, andfrom one nutrient medium to another. Hence, such a "natural" foamremoval procedure is not practical for most commercial purposes. Gravityseparation is accelerated by the addition of defoaming agent, and theuse of such agents in such a separation is sometimes practiced.

Control of the composition of solutes present in an aqueous fermentationliquid (so as thereby to minimize the presence of surface activecomponents therein) is difficult to achieve for many reasons. For onething, the exact composition of many nutrients and of metabolites frommany microorganisms is not now known.

The art of microorganism fermentation, particularly under high pressureconditions, has great need for a new and effective technique formechanically degassing a foamed fermentation broth.

SUMMARY

An object of the present invention is to provide a new, improved,effective, economical, and reliable process for degassing a previouslypressurized, foam-filled fermentation broth containing culturedmicroorganisms.

Another object is to provide a new, improved, economical, and reliablecontinuous process for accomplishing sequentially fermentation anddegassing of fermentation broths without using any defoaming agents andby using only a single pressurized fermentation zone and a single,separate degassing zone in generally adjacent relationship to saidfermentation zone.

Another object is to provide new and improved apparatus for use indegassing heavily foamed broth from a pressurized fermentor.

Another object is to provide new and improved apparatus for continuouslymaking defoamed fermentation broths which apparatus is suitable for usein pressurized fermentation with high gas flow and which can produce abroth containing a high content of cultured microorganisms without theuse of any defoaming agents.

Another object is to provide improved apparatus and methods fordegassing (defoaming) a pressurized fermentation broth, particularly onewhich is continuously produced using high gas flow, wherein the broth isfirst passed through an interstage cooler before being degassed.

Another object is to provide apparatus and technique for cooling apressurized, heavily foamed fermentation broth before degassing iscommenced.

Other and further objects, aims, features, advantages, purposes,applications, embodiments, and the like will be apparent to thoseskilled in the art from the teachings of the present specification takenwith the accompanying drawings.

The present invention is directed to improved technology for degassingfoamed fermentation broths, particularly pressurized such broths,containing cultured microorganisms, without the use of defoaming agentsand without appreciably damaging the cultured microorganisms.

Thus, in one aspect, the present invention provides a new and veryuseful method for degassing a fermentation broth which foamed (that is,a liquid broth containing a dispersed gas phase which is mainly in theform of bubbles) and which contains cultured microorganisms. Thestarting broth is at a temperature of from above about 20° to about 60°C. The method involves first the passing such broth while pressurized toat least about 3 psig into and through a cooling zone. In the coolingzone, the broth is cooled to a temperature ranging from 0° (e.g.freezing) to below about 20° C. The advantages of passage through such acooling zone are explained below.

Then the pressurized and cooled broth is subjected to degassing bypassing such through a first spray nozzle means. This nozzle meansdischarges a spray suspension of such broth into a gaseous spray zone(or chamber) located in a gravitationally upper part of a degassing zone(or chamber). The spray zone is vented to the atmosphere.

This nozzle means is adapted to pass particulate bodies having anaverage particle size of at least about 0.0005 millimeters and toproduce spray droplets having an average size of not greater than about2.5 millimeters. In general, the nozzle means is chosen so that theindividual discrete particles and/or droplets of the spray suspensionissuing therefrom provide exposure in the gaseous spray zone of amaximal surface area relative to the volume thereof, thereby to enhanceseparation of gas from the liquid phase of the fermentation broth. Thenozzle means, however, is also chosen so that, during the atomizing, noappreciable damage to, or hindrance to the passage therethrough of,microorganisms results. The nozzle size characteristics above indicatedappear to accomplish these desired results.

The spray suspension from this nozzle means is coalesced in thedegassing zone to form a bulk liquid phase located in a gravitationallylower portion thereof whereby the upper surface of such bulk liquidphase can be considered to form the bottom surface of such spray zone.

This liquid surface is characteristically covered by a layer of foamwhich itself has a surface. This nozzle means is so oriented that thespray suspension issuing therefrom is directed downwardly towards thesurface of the bulk liquid phase and the surface of the foam layerthereon. The action of the downwardly moving spray suspension instriking the surface of the foam layer causes destruction of this foamsurface and results in further release and separation of gas from theliquid phase of the fermentation broth.

A substantial percentage (typically more than about 50% by volume) ofthe total releasable (separatable) gas present in a starting foamedfermentation broth is characteristically separated from the liquid phaseof the fermentation broth by this sequence of spraying a directed spraysuspension and coalescing.

Preferably, but optionally, a portion of the bulk liquid phase iscontinuously withdrawn from a gravitationally low region of the bulkliquid phase, mechanically pressurized (as with a pump), and passedthrough a second spray nozzle means located in the spray chamber. Thissecond spray nozzle means is similar in function and characteristics tothe first spray nozzle means. The resulting second spray suspension islikewise downwardly directed towards the surface region of such foamlayer. This recycling procedure enhances the degassification of suchcoalesced bulk liquid phase not only through the action of the secondspraying and the coalescing thereof, but also through the action of thedirected recycle spray suspension striking the foam layer and causingsurface foam destruction.

The degassed fermentation broth is removed from a lower region of theliquid bulk phase collected in the degassing zone bottom region.

The degassing process can be practiced either batchwise or continuously(preferably the latter).

In another aspect, the present invention provides a new and very usefulcontinuous process for accomplishing direct preparation of a degassedfermentation broth of cultured microorganisms which broth is adapted tobe completely free from defoaming agents. In this process, water,nutrients, and air in respective selected amounts are chargedcontinuously to a pressurized fermenting zone which has been inoculatedwith a selected microorganism species. The fermenting zone is maintainedunder controlled conditions of temperature, pressure, agitation, anddilution rate. Under continuous operating condition, a heavily foamedfermentation broth is continuously removed from the pressurizedfermenting zone at a generally constant rate and directly charged firstto and through a beat exchange zone and then to a degassing zone(through first spray nozzle means thereof in the manner indicatedherein). Recycling of broth (through a second spray nozzle means) ispreferably practiced in the degassing zone in the manner indicatedherein. An effluent stream comprised of degassed product fermentationbroth is withdrawn continuously and at a generally constant rate fromthe degassing zone which rate is generally equal to the rate at whichfoam-filled fermentation broth is withdrawn from such fermenting zoneand charged to the degassing zone. The effluent stream withdrawn fromthe degassing zone can be used as desired. For example, the broth can bepasteurized and then spray dried.

In another aspect, the presentation provides a new degassed fermentationbroth having a high (in excess of about 100 grams per liter) content ofcultured microorganisms which is prepared directly and continuously in atwo-zone preparation sequence.

In still another aspect, the present invention is directed to new andvery useful improved apparatus for accomplishing microorganismfermentation broth degassing using a heat exchanger and degassingapparatus in combination.

In still another aspect, the present invention is directed to new andvery useful apparatus for continuous pressurized fermentation anddegassing using a sequentially interconnected combination of apressurized fermentor, a heat exchanger, and a degassing apparatus.

Because of the surprising effectiveness of the present improveddegassing technique and apparatus, process efficiency and economics forproducing microorganisms are raised to levels not heretofore attainable.

BRIEF DESCRIPTION OF DRAWINGS

In the drawings:

FIG. 1 is a diagrammatic representation partially in side elevation andpartially in vertical section illustrating (a) one embodiment ofdegassifier apparatus of this invention, and (b) methods for using thesame;

FIG. 2 is a simplified flow diagram illustrating one embodiment of acontinuous fermentation and broth degassing process of this invention;and

FIG. 3 is a simplified diagrammatic representation illustrating anapparatus configuration for practicing the process of FIG. 2;

DETAILED DESCRIPTION Microorganisms

The processes of the present invention can be utilized withmicroorganisms capable of producing nontoxic proteins. Suitablemicroorganisms include bacteria, yeasts, and molds. Yeasts are presentlypreferred.

Suitable yeasts include species from the genera Candida, Kluyveromyces,Hansenula, Torulopsis, Saccharomyces, Pichia, Pbaffia, Debaryomyces,Lipomyces, Crytococcus, Nematospora, and Brettanomyces. The preferredgenera include Candida, Kluyveromyces, Hansenula, Torulopsis, Pichia,Phaffia and Saccharomyces. Examples of suitable species of yeastinclude:

    ______________________________________                                        Candida boidinii   Hansenula saturns                                          Candida mycoderma  Hansenula californica                                      Candida utilis     Hansenula mrakii                                           Candida stellatoidea                                                                             Hansenula silvicola                                        Candida robusta    Hansenula polymorpha                                       Candida sake       Hansenula wickerhamii                                      Candida claussenii Hansenula capsulata                                        Candida rugosa     Hansenula glucozyma                                        Brettanomyces petrophilium                                                                       Hansenula henricii                                         Hansenula minuta   Pichia membranefaciens                                     Hansenula nonfermentans                                                                          Pichia pinus                                               Hansenula philodendra                                                                            Pichia pastoris                                            Torulopsis candida Pichia trehalophila                                        Torulopsis bolmii  Saccharomyces cerevisiae                                   Torulopsis versatilis                                                                            Kluyveromyces fragilis                                     Torulopsis glabrata                                                                              Saccharomyces rosei                                        Torulopsis molishiana                                                                            Bailii acidifaciens                                        Torulopsis nemodendra                                                                            Saccharomyces uvarum                                       Torulopsis nitratophila                                                                          Saccharomyces elegans                                      Pichia farinosa    Saccharomyces rouxii                                       Pichia polymorpha  Kluyveromyces lactis                                                          Phaffia rhodozyma                                          ______________________________________                                    

Suitable bacteria include species from the genera Bacillus,Mycobacterium, Lactobacillus, Leuconostoc, Streptococcus, Pseudomonas,Methanomonas, Brevibacterium, Acetobacter, Micrococcus, Corynebacterium,Achromobacter, and Methylobacter. Preferred genera include Bacillus,Pseudomonas, Protaminobacter, Lactobacillus, Leuconostoc, Streptococcus,Micrococcus, Arthrobacter and Corynebacterium.

Examples of suitable species of bacteria include:

    ______________________________________                                        Bacillus subtilus Pseudomonas ligustri                                        Bacillus cereus   Pseudomonas orvilla                                         Bacillus aureus   Pseudomonas methanica                                       Bacillus acidi    Pseudomonas fluorescens                                     Bacillus urici    Pseudomonas oleovorans                                      Bacillus mycoides Pseudomonas putida                                          Bacillus circulans                                                                              Pseudomonas boreopolis                                      Bacillus magaterium                                                                             Pseudomonas pyocyanea                                       Bacillus licheniformis                                                                          Pseudomonas methylphilus                                    Pseudomonas methanolica                                                                         Pseudomonas brevis                                          Pseudomonas acidovorans                                                                         Methylomonas agile                                          Pseudomonas methanoloxidans                                                                     Methylomonas albus                                          Protaminobacter ruber                                                                           Methylomonas rubrum                                         Methylomonas methanolica                                                                        Corynebacterium simplex                                     Mycobacterium rhodochrous                                                                       Leuconostoc bulgarions                                      Streptococcus cremoris                                                                          Lactobacillus bulgarions                                    Streptococcus lactis                                                                            Lactobacillus acidophilus                                   Streptococcus thermophilus                                                                      Corynebacterium alkanum                                     Leuconostoc citrovorum                                                                          Leuconostoc dextranicum                                     Corynebacterium   Mycobacterium phlei                                         hydrocarbooxydans Corynebacterium oleophilus                                  Mycobacterium brevicale                                                                         Nocardia salmonicolor                                       Corynebacterium   Nocardia minimus                                            hydrocarboclastus Nocardia corallina                                          Corynebacterium glutamicum                                                                      Nocardia butanica                                           Corynebacterium viscosus                                                                        Rhodopseudomonas capsulatus                                 Corynebacterium dioxydans                                                                       Microbacterium ammoniaphilum                                Corynebacterium alkanum                                                                         Archromobacter coagulans                                    Micrococcus cerificans                                                                          Brevibacterium butanicum                                    Micrococcus rhodius                                                                             Brevibacterium roseum                                       Arthrobacter rufescens                                                                          Brevibacterium flavum                                       Arthrobacter parafficum                                                                         Brevibacterium lactofermentum                               Arthrobacter simplex                                                                            Brevibacterium paraffinolyticum                             Arthrobacter citreus                                                                            Brevibacterium ketoglutamicum                               Methanomonas methanica                                                                          Brevibacterium insectiphilium                               Methanomonas methanaooxidans                                                  ______________________________________                                    

Suitable mold include species from the genera Aspergillus, Monilia,Rhizopus, Penicillium, Mucor, Alternaria and Helmintbosporium.

Examples of suitable species of molds include:

    ______________________________________                                        Aspergillus niger  Penicillium griseofulvum                                   Aspergillus glaucus                                                                              Penicillium expansum                                       Aspergillus flavus Penicillium digitatum                                      Aspergillus oryzae Penicillium italicum                                       Aspergillus terreus                                                                              Rhizopus nigricans                                         Aspergillus itconicus                                                                            Rhizopus oryzae                                            Penicillium notatum                                                                              Rhizopus delemar                                           Penicillium chrysogenum                                                                          Rhizopus arrhizus                                          Penicillium glaucum                                                                              Mucor mucedo                                               Rhizopus stolonifer                                                                              Mucor genevensis                                           ______________________________________                                    

Examples of suitable yeast species include Candida utilis, Saccbaromycescerevisiae, Kluyveromyces fragilis, Saccharomyces uvarum, Phaffiarhodozyma and Pichia pastoris. The most preferred microorganisms for usein the present invention include those yeasts which are currentlyapproved by the FDA for human consumption.

Fermentation

Fermentation (or microbial culture) of chosen or selected microorganismsis accomplished in a fermenter apparatus (or fermenting zone) using afermentation method.

The particular fermentation method and apparatus used to culture thechosen microorganism is not critical to the practice of the presentinvention. There are numerous fermentation processes and apparatusesthat are well known to those skilled in the art, both batch andcontinuous. Any of these well known fermentation processes andfermentors are suitable for use with the present invention, providedsuch is/are appropriate for the particular microorganism being cultured(as those skilled in the art will readily appreciate). It is preferredto use a freshly prepared, foamed fermentation broth in the practice ofthis invention.

Examples of suitable fermentors and fermenting processes are described,for example, in Perry's Chemical Engineer's Handbook Sixth Edition(copyright 1984) published by McGraw Hill, Inc., New York, New York atpages 27-5-27-13.

Typically microorganisms are cultured under aqueous phase conditions bygrowing them on a suitable nutrient composition which typically includesa carbon and energy source, an assimilable nitrogen source, mineralsalts, molecular oxygen (usually derived from air) with suitable pHmaintenance, and with other growth factors optionally being present, ifdesired, as those skilled in the art will readily appreciate.

Typically, the nutrient composition may vary depending upon such factorsas the microorganism species being cultured, nutrients available, andthe like. Selection of nutrients for a given such composition for use inculturing a particular microorganism species is known to those skilledin the art (see for example the "Manual of Methods for GeneralBacteriology," American Society for Microbiology, Washington, D.C., and"Yeast: Characteristics and Identification," Cambridge University Press,Cambridge, GB).

Suitable and presently preferred batch and continuous fermentationconditions are illustrated in Table I below:

                  TABLE I                                                         ______________________________________                                        Fermentation Conditions                                                                       Range                                                         Condition (or Variable)                                                                         Broad       Preferred                                       ______________________________________                                        Temperature (°C.)                                                                          20-60.sup.(6)                                                                             25-50.sup.(6)                                 Pressure (psig).sup.(3)                                                                            0-70       10-50                                         Retention Time (hour).sup.(1)                                                                      1-40        2-15                                         pH                   2-7         3-7                                          Agitation (rpm).sup.(5)                                                                           100-1200    400-800                                       Aeration (vvm).sup.(2)                                                                             2-7         3-5                                          Dilution Rate (h.sup.-1).sup.(1)(4)                                                             0.025-1     0.067-0.5                                       Total nutrients (dry wt.                                                                          50-700      100-400                                       grams per liter)                                                              ______________________________________                                         Table I footnotes:                                                            .sup.(1) Applies when continuous fermentation is carried out.                 .sup.(2) "vvm" indicates ratio of volume of air input (measured at            atmosperic pressure) to volume of fermentor broth.                            .sup.(3) "psig" indicates pounds per inch gauge.                              .sup.(4) "h.sup.-1 " indicates reciprocal hours.                              .sup.(5) Using a turbine bladed agitator or equivalent.                       .sup.(6) Where the upper temperature in any given fermentation is chosen      so as to be substantially nonlethal to the particular microorganisms issu     being cultured.                                                          

One presently preferred fermentation procedure for use in the practiceof the present invention is shown and described in Wegner U.S. Pat. No.4,617,274 entitled: "Biochemical Conversions by Yeast Fermentation atHigh Cell Densities," which teaches a pressurized fermentationtechnique, and which is incorporated by reference.

It is presently very much preferred to practice pressurized fermentationwhen using the degassing technique and apparatus of this invention. Forone thing, the need for a pump to pressurize a foamed fermentation brothis not needed since the pressure existing in the fermentor itself issufficient to transport effluent from the fermentor to the heatexchanger and then to the degasser (and the first atomizing nozzlethereof). This direct interconnection avoids the problems of trying topump or pressurize a foamed fermentation broth before feeding such tothe first atomizing nozzle.

Fermentation Broth

The product of a microbial fermentation procedure is conventionallytermed a fermentation broth. Such a broth is characterized by beingcomposed of:

spent culture medium (usually expressed on a dry solids bases)

dispersed gas (in the medium mainly as bubbles or foam)

cultured microbial cells (suspended in the medium)

fermentation by-products (mainly dissolved in the medium)

water

Broadly, a fermentation broth contains from about 5 to 180 grams perliter of harvestable cultured microorganisms (or microbial cells). Whenthe operating conditions of Table I are practiced, the productfermentation broth is characterizable as shown in Table II. Such a brothcomprises a starting system or material adapted for use in the practiceof the degassing technique of the present invention.

                  TABLE II                                                        ______________________________________                                        Fermentation Broth                                                                            Range                                                         Component (or variable)                                                                         Broad    Preferred                                          ______________________________________                                        Spent medium solids.sup.(3)                                                                      0-25     5-15                                              (dry weight, g/L,.sup.(2)                                                     degassed, spent broth)                                                        Cultured Microbial                                                                               5-180   100-160                                            cells (dry weight, g/L,                                                       degassed broth                                                                Fermentation       0-100    0-15                                              by-products (dry                                                              weight, g/L degassed                                                          broth)                                                                        Dispersed gas bubbles                                                                           20-60    40-55                                              (%, v/v).sup.(1)                                                              Water (g/L of degassed                                                                          100-900  300-850                                            broth)                                                                        ______________________________________                                         Table II footnotes:                                                           .sup.(1) % v/v indicates volume gas per unit volume of liquid multiplied      by 100 (e.g. volume percent of gas per liter of broth).                       .sup.(2) g/L indicates grams per liter.                                       .sup.(3) "Spent medium" includes any unconsumed nutrients.               

As can be seen from Table II, the preferred starting fermentation brothsare characterized by having a relatively high microbial cell content aswell as by having a relatively high dispersed gas content.

In this invention, the problem of separating gas from a starting brothhaving a high dispersed gas content is solved by the use of the new andvery useful degassing procedure and the associated apparatus.

Cooling

Introduction of a cooling zone (heat exchanger) between the fermentationzone and the degassing zone, as shown for example in FIG. 2, achieves aplurality of surprising advantages.

For one thing, the cooling of this gas-filled or foamed fermentationbroth maximizes the gas release and separation capability of thedegassing procedures and techniques taught herein. The cooling aids infoam breaking and enhances gas release.

For another thing, the cooling slows down the microorganisms' metabolicand physiological activities. For example, the microorganism respirationrate is reduced thereby, preferably to a very low rate. This activityreduction is desirable because further microorganism growth downstreamof the fermentor can cause production problems, such as, for example,the production of foam in the fermentation broth after degassing astaught herein.

For another thing, the cooling permits achievement of a size reductionin the dimensions of the degassing tank (see tank 11 in FIG. 1, forexample). Not only is the fermentation broth volume minimized thereby,but also the need for effervescensce of coalesced liquid in thedegassing tank (in order to achieve a desired level of residual gas) isreduced along with the volume of, and retention time for, coalescedliquid being held in such tank.

For another thing, the volume of the foam layer formed on the surface ofcoalesced liquid in the degassing tank is reduced. The effectiveness ofthe spray dispersions released from the atomizing nozzle means in thistank thus tends to be made more effective in reducing the percentage offoam present on the surface of such coalesced liquid.

Overall, the efficiency of degassing as taught by this invention isimproved as is the total volume of separated gas from degassedfermentation broth compared to a similar degassing procedure practicedwithout use of such a cooling zone.

In addition, the cooling zone permits one to avoid the need forjacketing on the degassing tank with attendant savings in equipment andoperational costs. In place of a cooling jacket about the degassingtank, a termally insulating blanket can be used, if desired. Residencetime of coalesced liquid in the degassing tank is preferably chosen sothat, under continuous, steady state operating conditions, nosignificant temperature increase in such liquid over the broth chargingtemperature occurs. If desired, a post-degassing zone cooling zone (notshown) can be utilized, if desired to maintain the degassed broth in amaximized chilled state before subsequent processing is undertaken.

Further, and particularly in the case where pressurized fermentation isbeing practiced, the need for a separate pump to move the effluent fromthe fermentor through the heat exchanger and into the degasser (astaught herein) is avoided. Fermentor pressures are preferably chosen soas to be adequate for such transport purposes. This is a desirableresult since pumping of a foaming fermentation broth can be difficult toaccomplish in a controlled and continuous manner. By using fermentorpressure as the transfer power, the flow rate of fermenation broth fromthe fermentor can be regulated easily and remotely by a suitableconventional throttle valve (or equivalent) located at or near the socalled "harvest port" at the bottom of the fermentor in the effluentline system.

Referring to FIGS. 2 and 3, and in accord with this invention it is seenthat a conventional heat exchanger 53 is inserted into aninterconnecting line from fermentor 49 to degassifier 51. The coolantfor the heat exchanger can be, for example, water, glycol, or mixturesthereof. Conventional coolant cooling apparatus (not shown) can be used.Control of the cooling of the fermentation broth passing through theheat exchanger and regulation of the temperature of the broth exitingfrom the heat exchanger is effected by modulation of the coolant beingconcurrently circulated through the heat exchanger (as distinct from thebroth). In practice, it is preferred to select a heat exchanger which islarge enough to accept various flow rates of coolant and of broth sothat a desired exit temperature for broth can be maintained throughcoolant flow rate (at some selected coolant temperature such as could beused during continuous operating conditions).

Conditions of cooling zone (heat exchanger) operation are summarized inTable III below:

                  TABLE III                                                       ______________________________________                                        Cooling Conditions                                                                             Range                                                        Condition (or Variable)                                                                          Broad       Preferred                                      ______________________________________                                        Temperature (°C.)                                                                         above 20-60°                                                                       25-50                                          Output Temperature (°C.).sup.(1)                                                          from 0-20°                                                                         from 0-5                                       Zone Residence Time (min)                                                                        0.05 to 5   .01 to l                                       Input Pressure (psig)                                                                            5 to 100 psig                                                                             10 to 50                                       Output Pressure    5 to 70 psig                                                                              10 to 50                                       ______________________________________                                         Table III footnotes:                                                          .sup.(1) A zero temperature here means "above freezing" as regards the        broth.                                                                   

Under preferred continuous operating conditions, a 10° C. temperaturereduction between input temperature and broth output temperature isdesirable for reasons of process economies and operating costreductions, as those skilled in the art will appreciate.

Degassification

Degassification of a fermentation broth, such as above characterized, iscarried out in accord with the teachings of the invention.

Referring to FIG. 1, there is seen one embodiment of degasser apparatusof this invention which is herein designated in its entirety by thenumeral 10. Degasser apparatus 10 incorporates a tank 11 which ispreferably fabricated of stainless steel, or of glass lined steel, or ofother materials which provide interior wall surfaces which aresubstantially inert to such materials as microorganisms, the brothholding the same, and the like. Preferably, the bottom wall surfaces 15of tank 11 are sloped (as shown) to a drain aperture or port 33 for easein draining fluid from tank 11, such as degassed, sprayed, coalesced andcollected liquid broth 12, and also the top wall surfaces 17 of tank 11are preferably dome configured (as shown), or conically tapered (notshown), for similar reasons and also for purposes of providing anoverhead gas or vapor space 13. Preferably, the tank 11 has a generallycylindrical mid-portion and is generally cross-sectionally circularrelative to a longitudinal axis and wherein said vessel has a ratio ofaverage diameter in said mid-portion to said longitudinal axis rangingfrom about 1:1 to 5:1.

If desired, the tank 11 may be provided with a jacket 14circumferentially extending around side wall portions thereof throughwhich a temperature regulating coolant liquid, such as water, glycol,mixtures thereof, or the like, can be circulated via input line 19 andoutput line 21 to control the temperature of liquid broth 12 in tank 11.However, it is one of the advantages of this invention usage of thejacket 14 can be avoided by use of the heat exchanger 53 as shown inFIG. 2 and FIG. 3. Also, tank 11 is preferably provided with a sightglass 16 which is conveniently interconnected with a location in bottomwall surfaces 15 and with a location in top wall surfaces 17 by pipes18, thereby permitting an operator to observe the liquid level in tank11 during operation of degasser apparatus 10. If desired, an observationwindow (not shown) can be placed at a location in top wall surface 17.

Through a central upper location in top wall surfaces 17 an input pipe22 extends which terminates in space 13 in an associated atomizingnozzle 23. Preferably, the pipe 22 slidably extends through a sleeve 24that is fixed by welding or the like to adjacent top wall surfaces 17and pipe 22 is thus slidably extendable or retractable through sleeve 24so as to permit the spatial location of nozzel 23 relative to the uppersurface 26 of liquid 12 to be adjustable. A set screw 27 or the likethreadably mounted through sleeve 24, or some other equivalentarrangement, is used to clamp pipe 22 in a desired set position. Theouter end 28 of pipe 22 is functionally associated with a flexible tube29 by coupling means (not shown), and tube 29 similarly joins an inputfeed pipe 30. More than one input nozzle can be provided if desired. Inoperation of degasser apparatus 10, a pressurized gas-bubble containing(or foaming) fermentation broth is charged into tank 11 through thesequence of tube 29, pipe 22, and nozzle 23. The atomized fermentationbroth in space or chamber 13 condenses and/or coalesces in tank 11 toform collected liquid broth 12 in a lower portion of tank 11. Theatomization resulting from passage of the fermentation broth throughnozzle 23 functions as a foam-breaking technique. The vapor space 13 ismaintained at ambient (atmospheric) pressure by vent means associatedwith from vapor space 13, the vent means here being illustrativelyprovided by vent pipe or duct 31 associated with an upper centrallocation of top wall surfaces 17. If desired, and as shown, an exhaustfan 45 is associated, for example, with an upper end portion of duct 31to assure the maintenance of a positive air flow therethrough and anambient pressure in space 13 during operation of apparatus 10.

In practice, it is found convenient and preferable to maintain theposition of liquid surface 26 relative to the side walls of tank 11 suchthat surface 26 falls in the range from about 1/5 to 4/5 of the distancefrom the top most interior location of tank 11 (relative to the totalinterior height of tank 11); however, any convenient liquid level can beused. Characteristically, surface 26 is covered by a layer 25 comprisedof foam. Not only does the tank 11 thus serve as a holding tank adaptedto accomodate an emergency or temporary interruption in a degassingprocess of this invention, such as hereinbelow described, but also thetank 11 serves to maintain a quantity of collected liquid broth 12 intank 11 from whose surface 26 residual quantities of gas can escape byeffervesence, which is desirable.

The nozzle 23 is oriented (or directed) towards the surface 26. Thespray suspension issuing from the oriented nozzle 23 is directed againstthe upper surface 35 of the foam layer 25 which serves to cause a breakup of surface foam cells in foam layer 25, which is desirable.

In operational practice, it is preferred to provide degasser apparatus10 with an optional recycle loop which is designated in its entirety bythe numeral 32. While many different apparatus configurations forrecycle loop 32 are possible, as those skilled in the art will readilyappreciate, the apparatus 10 embodiment is provided with a recycle loop32 which comprises in connected succession line 34, pipe 38, flexibletube 39, pipe 41, and second atomizing nozzle 42. The subassembly oftube 39, pipe 41, and nozzle 42 is similar to the above describedsubassembly of tube 29, pipe 22, and nozzle 23 in structure andoperation; thus, pipe 41 slidably adjustably moves and extends throughsleeve 43 with set screw 44 mounted in sleeve 43 being used to positionand clamp pipe 41. More than one recycle loop or recycle nozzle meanscan be used if desired. In apparatus 10, the piping arrangement used isthus such that only a single pump 36 is used to remove liquid broth fromtank 11 both for discharge of effluent (as for further processing) andfor recycle. If desired, alternatively, one pump can be used foreffluent control and a second pump can be used for recycle control, forexample. An adjustable valve 46 in line 38 is conveniently provided toaid in regulating the recycle rate. Also, flow regulating valves 47 and48 can optionally be provided in line 34 between pump 36 and pipe 38,and in discharge line 37 after line 38, respectively.

Thus, if recycling is practiced, the recycle rate can be independentlyregulated separately from the discharge rate, if desired. The recycleloop 32 is found to enhance the separation of gas from fermentationbroth particularly when pressurized fermentation broths are continuouslycharged to the degassing zone. Use of the recycle loop 32 is preferredwhen it is desired to separate as much as practical of the gas presentin a fermentation broth entering tank 11 through line 30, tube 29, pipe22, and nozzle 23. The recycle loop 32 is particularly preferred whenfoam-filled fermentation broth from a pressurized fermentor is beingcontinuously charged to degasser apparatus 10.

Collected liquid broth 12 which has been degassed in apparatus 10 isremoved from tank 11 through the port 33. Port 33 feeds into take-offline 34. Conveniently, line 34 is equipped with a pump 36 which has thecapacity to produce adjustable flow rates and adjustable outputpressures (such as a variable speed centrifugal pump), therebypermitting the rate and the pressure at which liquid broth 12 iswithdrawn from tank 11 to be regulated and controlled in a desiredmanner. Between port 33 and pump 36, an adjustable throttle valve 40 orthe like is located for purposes of regulating effluent removal fromtank 11 through port 33. Instead of being recycled, effluent from pump36 can pass into line 34 and then into discharge line 37 which leads todownstream processing zones or stations. For example, effluent fromdischarge line 37 can be pasteurized to kill the microorganismscultured, and then the resulting effluent can be spray dried to recoverdried cultured microorganism residues, as those skilled in the art willappreciate.

As those skilled in the art will appreciate, the degasser apparatus anddegassing process of this invention can be employed for degassingfermentation broths of widely different composition under widelyvariable entering pressures, whether continuous or batch operatingconditions are contemplated. A non-pressurized fermentation broth can bepressurized by a feed pump and then passed through nozzle 23. For lowcharging pressures (such as under about 10 lbs/in² gauge (psig)) and lowgas volumes (under about 20% V/V), a single stage of atomization usingthe tank 11 may be sufficient without utilization of a recycle loop toeffectuate a desired level of gas removal from entering or startingfermentation broth. Use of a combination of two stages of atomization ina single degassing tank (one stage being atomization of the input streamand another stage being the atomization of the recycle stream) as taughtherein has been found capable of achieving substantially completedegassing of a pressurized fermentation broth, depending upon conditionsand circumstances. For example, a starting broth under a pressure of 40psig or higher, and having a total solids content (dissolved andsuspended) of greater than about 7.5 weight percent, and having a gascontent of greater than about 50 volume percent (all based on totalfermentation broth) can be degassed; however, substantially higher orlower operating degasser input pressures, solids contents, and gascontents appear to be feasible with suitable degasser apparatusembodiments of this invention.

While the vapor space 13 is preferably maintained at atmosphericpressures during operation of apparatus 10, as indicated, those skilledin the art will appreciate that the vapor space 13 can be maintained atsubatmospheric pressure or super atmospheric pressure if desired duringsuch operation. However, subatmospheric pressures now appear to havetendency to cause operating problems apparently caused by excessive foamproduction, while super atmospheric pressures appear to result in higheroperational costs since depressurizing to atmospheric pressures is onlypostponed to a subsequent stage, such as a second degassing stage. Asecond degassing stage connected in series with a first degassing stageappears to have no functional or processing advantage at this time.Thus, apparatus 10 is useful for degassifying fermentation broths eitherproduced at ambient or low pressures or produced at elevated pressures.Continuous operating conditions are preferred.

Illustrative degassing apparatus operating conditions adapted fordegassing in accord with the present invention are shown in Table IVbelow:

                  TABLE IV                                                        ______________________________________                                        Degassification Process Parameters                                                              Ranges                                                      Condition (or Variable)                                                                           Broad      Preferred                                      ______________________________________                                        Nozzle size (mm)    0.0005-2.5.sup.(1)                                                                       0.001-3.sup.(1)                                First Nozzle pressure (psig)                                                                         3-100      5-50                                        Starting temper. °C..sup.(2)                                                                  20-60     25-50                                        Interior temper. °C..sup.(3)(4)                                                               0-60       4-50                                        Second Nozzle pressure (psig)                                                                        1-50       5-45                                        ______________________________________                                         Table IV footnotes:                                                           .sup.(1) The lower value indicates the average minimal particle size          presented in suspended form in a starting fermentation broth which can be     passed through a nozzle orfice, while the upper value indicates the           average maximum droplet size which is produced after passage of a startin     fermentation broth through an atomizing nozzle.                               .sup.(2) The starting temperature referenced is the temperature of a          fermentation broth entering the first spray nozzle in the degasser            apparatus (from the fermentor).                                               .sup.(3) The interior temperature referenced is the interior temperature      of the degassing zone (both the liquid phase and gas chamber).                .sup.(4) A zero temperature here means "above freezing" as regards the        broth.                                                                   

Characteristically, by the practice of the present invention, one canobtain at least about a 95% reduction in gas volume in a fermentationbroth compared to a starting foamed fermentation broth (such as one froma pressurized fermentor operated with high air circulation) containingat least about40% by volume of gas.

Illustrative conditions for practicing the presently most preferredcontinuous high pressure fermentation cooling, and degassing process ofthis invention are provided in Table V below. The fermentation brothproduced by the Table IV continuous fermentation conditions hascharacteristics as set forth in the "preferred" ranges shown in Table IIabove.

                  TABLE V                                                         ______________________________________                                        Operating Process Conditions for High Pressure                                Continuous Fermentation and Degassing                                         Process Variable      Range                                                   ______________________________________                                        (Fermentation)                                                                pressure (psig)       20-50                                                   temperature (°C.)                                                                            20-40                                                   agitation (rpm)       500-800                                                 ammonia input (g/g cells)                                                                           0.8-1.2                                                 air input (vvm) (40 psig base)                                                                      3-6                                                     dilution rate (h.sup.-1)                                                                            0.17-0.25                                               retention time (hr)   4-6                                                     water                 to chosen level                                         total nutrients (dry wt., gas free H.sub.2 O)g/L                                                    100-400                                                 (Cooling)                                                                     output temperature (°C.)                                                                     0-5                                                     residence time (min.) less than 0.5                                           (Degassification)                                                             charging pressure (psig)                                                                             5-40                                                   chamber gas pressure (psig)                                                                         0.8-1.2                                                 recycle flow rate (gpm)                                                                             0.5-30                                                  recycle charging pres. (psig)                                                                        5-10                                                   chamber temperature (°C.)                                                                     4-10                                                   discharge rate (gpm)  0.5-30                                                  ______________________________________                                    

As those skilled in the art will appreciate, the broth retention time inthe degassifier is variable over a very wide range depending upon manyfactors, especially the growth rate of the particular microorganismbeing cultured. The fermentor temperature conditions shown in Table V,as those skilled in the art will appreciate, are particularly suitable,for instance, for yeasts. Bacteria, for example, can be cultured athigher temperatures, such as 70° C. or even higher. Also, the coolingtemperature of 0 referenced in Table V here means "above freezing".

The degassed fermentation broth produced by the process summarized inTable V characteristically bas more than about 90% of the gas present inthe starting heavily foamed fermentation broth separated therefrom andcontains more than about 100 grams per liter of cultured microorganisms.

Referring to FIGS. 2 and 3, there is seen one embodiment of a preferredintegrated continuous process for effecting fermentation ofmicroorganism under high pressure followed by cooling and then followedby degassing of the resulting foam-filled fermentation broth. Apparatusfor practicing this process is shown in FIG. 3. The process andapparatus employs a single fermentation zone, a single immediatelysucceeding heat exchange zone, and a single immediately succeedingdegassing zone. In the cooling zone and in the degassing zone, thedegassing techniques and apparatus of this invention are utilized. Inthe fermentation zone, the fermenting techniques and apparatus known tothe prior art are utilized. For example suitable fermentation apparatusis described in Wegner and Hunt U.S. Pat. No. 4,670,397 the disclosureof which is hereby incorporated by reference.

Prior to the present invention, so far as now known, no alternativedegassing technique and apparatus were known which were suitable forachieving the objectives of (a) accomplishing the desired and preferredhighly efficient continuous high pressure fermentation with high airflow and resultant production of a high solids content fermentationbroth, (b) subsequently accomplishing a substantially complete cessationof fermentation by a cooling procedure only, (c) subsequentlyaccomplishing the desired preferably substantially complete degassing ofa foam-filled fermentation broth without using any chemical defoamingagents, (d) using only a single continuously operating fermenting zonesequentially directly coupled first to a single continuously operatingcooling zone and then to a single continuously operating degassing zone,and (e) fermenting, cooling, and degassing without causing any damage tothe cultured microorganisms.

In practicing the continuous process of FIG. 2, using the apparatus ofFIG. 3, as those skilled in the art will appreciate, the microorganismis preliminarily inoculated into the fermentor (having been suitablycultured in auxiliary apparatus). In a start up procedure, the fermentoris gradually brought up to the operating conditions such as shown inTable IV. Thereafter, the effluent from the fermentor is continuouslyand directly charged into the degassifier which is operated under theconditions shown in Table IV.

In FIGS. 2 and 3, the fermentor 49, duly inoculated with a chosenmicroorganism for culturing, is continuously charged with oxygen,nutrients and water, for example, a diluted aqueous sucrose stream, anaqueous nutrient/salt stream, an aqueous trace mineral stream, a gasstream comprised of a compressed air suitably controlled by dissolvedoxygen monitoring in the fermentor, a gas stream comprised of compressedammonia controlled by internal fermentor pH, (both gas streams beingcharged by sparging). The conditions of fermentation are selected so asto maintain in the fermentation zone no excess sucrose yet the amount ofsucrose supplied is equal to the maximum effective amount which themicroorganisms can consume (metabolize). If an excess sucrose were to beallowed, then alcohol would be produced as a by-product which isundesirable. On the other hand, if less than the maximum usable amountof sucrose is supplied relative to the microorganisms being cultured,then maximum microorganism productivity is not achieved.

The effluent from the fermentor unit 49 is conveyed (moved) by a conduitdirectly and continuously to the heat exchanger unit 53 and then to thedegassification unit 51 and atomized (sprayed) therein as taught herein.After the coalesced liquid level in the degassifier reaches a desiredoperating height, as at the end of a start up procedure, then a recycleprocedure is started and maintained in the degasser unit. Conditions areshown in Table IV.

Particularly under the continuous operating condition shown in Table IV,it bas been discovered that, unless the heat exchange unit is presentbetween the fermentor and the degasser, the microorganisms continue togrow and emit heat even as the fermentor 49 effluent is moved to thedegasser 51. The result is that swings in the temperature of thecollected broth 12 in the tank 11 (referring to FIG. 1) can occur,causing thermal loads or difficulties in maintaining uniform heatcontrol of the broth 12 using the jacket 14. In order to overcome andobviate this problem, it bas been further discovered that the problemthus generated can be surprisingly overcome completely by inserting aninterstage heat exchanger 53 between the fermentor 49 and thedegassifier 51 resulting in a flow diagram as shown in FIG. 2 and anapparatus configuration as shown in FIG. 3. With the heat exchanger 53,the necessity for a jacket 14 is eliminated if desired.

EXAMPLES

The following examples are presented in further illustration of theinvention and are not to be considered as unduly limiting the scope ofthe invention.

Example 1 Continuous Fermentation

In a continuous aerobic fermentation process, dilute aqueous sucrose,dilute aqueous mineral salts, and compressed air and anhydrous ammoniawere charged to a 1500 liter capacity fermentor that had been inoculatedwith the yeast Candida utilis NRRL Y-1082 and the fermentor was operatedunder a pressure of about 40 psig and a constant weight was maintainedin the fermentor by automatic fermentor weight control so that thevolume of the foam-filled fermentation broth in the fermentor was about720 liters.

The pH was maintained automatically at about 4.0 by the ammoniaaddition. The temperature in the fermentor was maintained at about 35°C. by cooling coils in the fermentor and jacket around the outside wallsof the fermentor. The pressure was maintained automatically bycompressed air sparging into the fermentor. The air aeration rate wasabout four volumes of air (charged at about 60 psig and about 25° C.)per volume of fermentor broth per minute. Agitation sufficient tomaintain the fermentor broth in the reactor in a substantially uniformstate was accomplished by two paddle-type turbines mounted on a commonshaft in axially spared relationship to one another, the shaft beinggenerally vertically disposed in the fermentor along the fermentor axis,and the shaft being driven at about 800 rpm by an external powerhead.

Under steady-state conditions, foam-filled fermentor broth wascontinuously withdrawn from a bottom part in the fermentor through acontrollable harvest valve. It was found that the high gas content ofthe so-withdrawn effluent made such difficult to transport by pipe usinga pump.

The gas content (foam derived) of the effluent fermentor broth was notexactly known, but, from the following tests, it is estimated that thegas volume of the effluent broth was generally in the range of 40-60%v/v measured at atmospheric pressure:

Test a: An aliquot of the fermentation broth was removed from a samplingvalve located in the fermentor wall amount about 1/3 of the way up fromthe bottom thereof and its volume was immediately measured and found tobe 1400 mL. To this sample, silicone-based antifoam (4 drops) was addedfollowed by gentle agitation to ensure a uniform mixing of saidantifoam. After standing at about 25° C. for 4 minutes, the total liquidvolume of the resulting sample was found to be 780 mL. The foam (gas)volume was then calculated as 620 mL or 44.3% of the fermentation broth.

Test b: An aliquot of the fermentation broth in the effluent stream wascollected from the region of the harvest valve. The total broth volumewas immediately measured and found to be 1300 mL. To this fermentationbroth, 4 drops of an antifoam was added as in test (a) followed bygentle agitation. After standing at about 25° C. for four minutes, thetotal liquid volume was found to be 680 mL. The foam (gas) volume wasthen calculated as 620 mL or 47.7% of the fermentation broth dischargedfrom the harvest valve.

The effluent stream from the harvest valve of the pressurized fermentorwas continuously fed directly through a conduit to and through a heatexchanger which cooled the broth to a temperature of about 3° C. fromthe fermentor temperature of 35° C. By regulation of coolant flowthereafter, the chilled broth was immediately fed to a degasserapparatus of the type shown in FIG. 1 and charged through the firstspray nozzle thereof. The nozzle was able to pass particulates having anaverage particle size down to about 0.0005mm and was able to developspray droplets not larger than about 2.5mm. The nozzle used waspurchased commercially from the Spraying System Company under the tradedesignation "Fulljet 1/2 GG-25." The spray suspension from the nozzlewas directed downwardly towards the surface of the foam layer whichcharacteristically appeared on the surface of the coalesced liquidcollected in the lower regions of the degasser apparatus. Undercontinuous operating conditions, with no recycling, the nozzle head wasspaced at a distance of about 50 centimeters from the surface of thefoam layer, and a degassed effluent stream was continuously removed fromthe degasser apparatus at a rate sufficient to maintain a constant levelof coalesced liquid in the degasser apparatus (which was also aboutequal to the rate at which the liquid phase of the fermentor broth wasbeing withdrawn through the harvest valve). The temperature of thecoalesced liquid in the degasser apparatus was found to be about 5° C.with the cooling being exclusively from the heat exchanger and with nocirculation of glycol/water coolant in the jacket 14 of apparatus 10(FIG. 1).

Under substantially steady-state continuous operating conditions, analiquot sample (1300 milliliters immediately measured) such effluentstream from the degasser was collected. To this sample, four drops ofthe antifoam agent used in Tests (a) and (b) above were added followedby gentle agitation. After standing at about 25° C. for four minutes,the total liquid volume of the resulting sample was found to be 1160 ml.The calculated foam or gas volume of the thus collected broth was 140 mLor 10.8% of total broth volume. This represents a gas separation of morethan 75% by gas volume, compared to the results shown by test(b) above.

When the effluent from the degassifier apparatus was examined under amicroscope, it was found that the cultured microorganisms haveexperienced no noticable deterioration or physical damage as a result ofthe foregoing degassing procedure (compared to a correspondingexamination of the cultured microorganism, present in the effluent fromthe fermentor.

Example II

The procedure of Example I was continued except that recycle ofcoalesced liquid in the degasser apparatus was undertaken using therecycle loop (32) as in apparatus 10 of FIG. 1. The rate of continuousrecycle was chosen to be about equal to the rate at which effluent wascontinuously withdrawn from the degasser apparatus. The second atomizingnozzle was the same as the first atomizing nozzle (above described inExample I) and such second nozzle was likewise spatially oriented sothat the spray suspension produced therefrom was directed toward thesurface of the foam layer. The distance between the nozzle and the foamlayer was set at about 50 cm. The pressure before the second nozzle wasabout 5 psig (achieved by operation of pump 36).

Under steady state continuous operating conditions, with such recycle,an aliquot (1500 ml immediately measured) was separated from thecontinuous effluent stream being removed from the bottom of the degasserapparatus. Using the same procedure as described above in Example I forfoam measurement, it was found that the calculated foam (gas) volume was40 mL or 2.7% of the broth volume. This represents a reduction of about95% in gas volume when compared to the result obtained in Example I (b)above. When the effluent from the degassifier apparatus was examined,under a microscope, it was found that the cultured microorganisms haveexperienced no noticable deterioration or physical damage as a result ofthe foregoing degassing procedure (compared to a correspondingexamination of the cultured microorganism, pres(int in the effluent fromthe fermentor.

Example III

The following fermentation procedure is believed to be typical andillustrative of continuous fermentation of microbes:

    ______________________________________                                         5 to 35       parts by weight sucrose                                         1 to 20       parts by weight mineral salts                                  45 to 94       parts by weight water.                                         ______________________________________                                    

An inoculum of C. utilis yeast is added to this mixture.

The fermentor is sealed, agitation is started (twin paddle-type turbineson a single shaft rotating at about 600 rpm), and air is sparged intothe system at 25° C. at the rate of about 45 psig so that the aerationrate is about 3 volumes per volume of liquid ferment per minute. Also,ammonia is introduced at a rate sufficient to maintain pH at about 4.Fermentor temperature is maintained at about 35° C. through heatexchange coolant circulation.

The product broth is charged into a degasser apparatus similar to thatused in Example I with recycle and it is found that most of the gas inthe broth is separated from the broth liquid. No adverse effects on themicroorganisms are observed.

Example IV Batch Fermentation

Yeast grown in a batch mode in a 1,500 liter fermentor is used for thiscalculated illustrative embodiment. Initially, the duly-sterilized1,500liter fermentor, is loaded with 400 Kg of feed, and then isinoculated with 12 1 of inoculum containing about 1.2×10¹⁴ of the sameC. utilis yeast species indicated in Example I. The inoculum is grown ina small bench top fermentor. The inoculated 1500 liter fermentor isoperated aerobically at about pH 4.0 and about 35° C. until all of thecarbon source (sucrose) is consumed. Sterile additional nutrient feed isthen continuously added at a rate such that the culture is able tocompletely consume the carbon source as added, as monitored by dissolvedoxygen response. The air added is sterilized by filtration and spargedinto the liquid fermentor at a rate of about 2 volumes per volume ofgaseous ammonia is used to control the pH and provides the nitrogensource. When the fermentor weight reaches 800 Kg, the feed isdiscontinued and the whole fermentor content is discharged through theharvest value for degassing directly into a degasser apparatus similarto that used in Example I.

Recycle is employed. The coalesced liquid removed from the degasserbottom contains very few gas bubbles and the cultured microorganismsappear to show no effects caused by the degassing procedure.

While this invention has been described in detail for the purpose ofillustration, it is not to be construed as limited thereby, but isintended to cover all changes and modifications within the spirit andscope thereof.

What is claimed is:
 1. A process for degassing a starting fermentationbroth containing (a) a dispersed gas phase in the form of bubbles and(b) suspended cultured microorganisms and having a temperature rangingfrom above about 20° C. to about 60° C., said process comprising thesteps of(a) providing a degassing apparatus comprising: a generallyfluid tight vessel defining a degassing zone, said degassing zone havinga gravitationally upper portion and a gravitationally bottom portion;first spray nozzle means in flow communication with said upper portionand constructed so as to pass particulate bodies having an averageparticle size of at least about 0.0005 millimeters and to produce spraydroplets having average sizes not greater than about 2.5 millimeters;vent means in flow communication with said upper portion; and recyclemeans providing flow communication between said bottom portion and saidupper portion and including second spray nozzle means in flowcommunication with said upper portion and constructed so as to passparticulate bodies having an average particle size of at least about0.0005 millimeters and to produce spray droplets having average sizesnot greater than about 2.5 millimeters; (b) pressurizing a startingfermentation broth containing a dispersed gas phase in the form ofbubbles and suspended microorganisms to a pressure of at least about 3psig; (c) passing said pressurized broth through a heat exchange zone tocool said broth to a temperature ranging from above 0° C. to below about20° C; (d) charging said fermentation broth through said first spraynozzle means so as to form a first spray suspension, said first spraysuspension having a bottom surface; (e) coalescing said first spraysuspension to form a bulk liquid phase in said bottom portion of saiddegassing zone, said bulk liquid phase having a gravitationally upperportion, a gravitationally bottom portion, and an upper surface wherebysaid upper surface of said bulk liquid phase forms the bottom surface ofsaid first spray suspension; (f) directing said first spray suspensiondownwardly toward said upper surface; (g) removing a first portion ofsaid bulk liquid phase from said bottom portion of said degassing zone;(h) discharging said first portion of said bulk liquid phase throughsaid second spray nozzle means so as to form a second spray suspension;and (i) coalescing said second spray suspension into said bulk liquidphase, said second spray suspension being directed toward said uppersurface.
 2. A process of claim 1 wherein said pressure ranges from about5 to 50 psig while maintaining said starting fermentation broth at atemperature ranging from about 20° to 50° C.
 3. A process of claim 2wherein said broth is cooled to a temperature ranging from about 0 toabout 5° C. during said passing.
 4. A process of claim 3 herein a secondportion of said bulk liquid phase is continuously removed and separatedfrom said gravitationally bottom portion thereof.
 5. A process of claim4 wherein, after said upper surface initially reaches a level within apredetermined range in said vessel, said charging and said secondportion removing are each carried out at approximately constant andequal rates.
 6. A process of claim 5 wherein said first portion isremoved, pressurized, and discharged through said second nozzle means ata rate such that said second portion which is removed and separated isat least about 95 percent by volume free from the dispersed gas phaseassociated with said starting fermentation broth measured at atmosphericpressure.
 7. The process of claim 6 wherein said first portion and saidsecond portion are simultaneously withdrawn as a single common streamfrom a common location in said bottom portion of said degassing zonewhich common stream is then divided into said first portion and saidsecond portion.
 8. The process of claim 1 wherein interior portions ofsaid vessel are maintained at a temperature ranging from about 0° to 60°C.
 9. The process of claim 1 wherein said starting fermentation broth iscomprised of;(a) from 0 to about 25 grams per liter of degassedfermentation broth on a dry weight basis of spent medium solids, (b)from about 5 to 180 grams per liter of degassed fermentation broth on adry weight basis of cultured microbial cells, (c) from 0 to about 100grams per liter of degassed fermentation broth on a dry weight basis forfermentation by-products, (d) from about 20 to 60 volume % of gas perliter of said starting fermentation broth, and (e) from about 100 to 900grams per liter of degassed fermentation broth of water.
 10. The processof claim 1 wherein said starting fermentation broth is prepared in afermentation zone at a temperature of from about 20° to 60° C., providedthe upper temperature in any given fermentation is chosen so as to besubstantially non-lethal to the particular microorganisms beingcultured, at a pressure of from 0 to about 70 psig, at a pH of fromabout 2 to 7, while charging to said fermentation zone from about 2 to 7volumes of air per fermentation zone volume per minute and whilemaintaining in said fermentation zone a turbine-bladed agitatoroperating at from about 100 to 1200 rpm.
 11. A continuous process forthe direct preparation of a degassed fermentation broth which containsan excess of about 100 grams per liter of cultured microorganisms andwhich is substantially free from defoaming agents, said processcomprising the steps of continuously and sequentially:(a) providing afermentation apparatus defining a fermenting zone, said fermenting zoneincluding a growing microorganism species; (b) charging into saidfermenting zone; (1) water in an amount sufficient to maintain apredetermined liquid level in said fermenting zone, (2) from about 100to 400 grams of total nurient materials on a dry weight basis per literof gas-free water, (3) from about 3 to 6 volumes of air per volume ofsaid fermenting zone per minute based upon 40 psig; (c) maintaining insaid fermenting zone a dilution rate of from about 0.17 to 0.25 perhour, a pressure of from about 20 to 50 psig, a temperature of fromabout20° C. to 40° C., and at least sufficient agitation to keep thematerials in said fermentation zone in a substantially uniformly mixedcondition; (d) removing at said temperature and said pressure from saidfermenting zone the resulting foamed fermentation broth, said brothbeing comprised as follows: (1) from about 300 to 850 grams of water perliter of degassed broth, (2) from about 100 to 160 grams of dry weightmicroorganisms per liter of degassed broth, (3) from about 5 to 15 gramsof spent broth solids per liter of degassed broth, (4) from 0 to about15 grams of dry weight fermentation by-products per liter of degassedbroth, and (5) from about 40 to 55 volume percent of disperse, discreetgas bubbles; (e) providing a heat exchanger apparatus defining a heatexchanger zone; (f) passing said fermentation broth through said heatexchanger zone to cool said broth to a temperature in the range from 0°C. to about 20° C.; (g) providing a degassing apparatus comprising: agenerally fluid tight vessel defining a degassing zone, said degassingzone having a gravitationally upper portion and a gravitationally bottomportion; first spray nozzle means in flow communication with said upperportion and constructed so as to pass particulate bodies having anaverage particle size of at least about 0.0005 millimeters and toproduce spray droplets having average sizes not greater than about 2.5millimeters; vent means in flow communication with said upper portion;and recycle means providing flow communication between said bottomportion and said upper portion and including second spray nozzle meansin flow communication with said upper portion and constructed so as topass particulate bodies having an average particle size of at leastabout 0.0005 millimeters and to produce spray droplets having averagesizes not greater than about 2.5 millimeters; (h) charging said foamedfermentation broth through said first spray nozzle means so as to form afirst spray suspension, said first spray suspension having a bottomsurface; (i) coalescing said first spray suspension to form a bulkliquid phase in said bottom portion of said degassing zone, said bulkliquid phase having a gravitationally upper portion, a gravitationallybottom portion, and an upper surface whereby said upper surface of saidbulk liquid phase forms the bottom surface of said first spraysuspension; (j) directing said first spray suspension downwardly towardsaid upper surface; (k) removing a first portion of said bulk liquidphase from said bottom portion of said degassing zone; (1) dischargingsaid first portion of said bulk liquid phase through said second spraynozzle means so as to form a second spray suspension; (m) coalescingsaid second spray suspension into said bulk liquid phase, said secondspray suspension being directed toward said upper surface; and (n)removing and separating a second portion of said bulk liquid phase.